Benjamin Stodart

Genetic characterization of a novel Phomopsis sp., a putative biocontrol agent for Carthamus lanatus

A large number of isolates of Phomopsis sp. have been collected from the weed Carthamus lanatus (saffron thistle) in Australia, and their potential as biological control agents for weeds of the Asteraceae has been demonstrated. An analysis of their genetic diversity and a multigene phylogenetic analysis were undertaken to ascertain whether these isolates were distinct from other species of Phomopsis that commonly attack crop species in Australia. Minimal variation was found between the Phomopsis spp. isolated from saffron thistle, except two isolates that appeared to share identity with Diaporthe helianthii and P. viticola. Analysis of the selected isolates from saffron thistle with the nucleotide sequence of the partial ITS and tef1-α regions demonstrated that the sequences were distinct from all other species of Phomopsis so far described from crops in Australia. These findings provide strong support for the recognition of these isolates as a separate species of Phomopsis. The implications of these findings are discussed in relation to biological control of saffron thistle.

Development of a Genomics-Based LAMP (Loop-Mediated Isothermal Amplification) Assay for Detection of Pseudomonas fuscovaginae from Rice

The vast amount of data available through next-generation sequencing technology is facilitating the design of diagnostic marker systems. This study reports the use of draft genome sequences from the bacterial plant pathogen Pseudomonas fuscovaginae, the cause of sheath brown rot of rice, to describe the genetic diversity within a worldwide collection of strains representing the species. Based on a comparative analysis with the draft sequences, primers for a loop-mediated isothermal amplification (LAMP) assay were developed to identify P. fuscovaginae. The assay reported here reliably differentiated strains of P. fuscovaginae isolated from rice from a range of other bacteria that are commonly isolated from rice and other plants using a primer combination designated Pf8. The LAMP assay identified P. fuscovaginae purified DNA, live or heat-killed cells from pure cultures, and detected the bacterium in extracts or exudates from infected host plant material. The P. fuscovaginae LAMP assay is a suitable diagnostic tool for the glasshouse and laboratory and could be further developed for in-field surveys.

Polyphasic identification of Pseudomonas fuscovaginae causing sheath and glume lesions on rice in Australia

Pathogenic fluorescent pseudomonads associated with sheath brown rot disease symptoms on near-mature rice were characterised by a polyphasic study. Twelve strains of Pseudomonas fuscovaginae from the International Collection of Microorganisms from Plants (ICMP), which represent those strains lodged in several world culture collections, were used for comparison. Two strains, ICMP 9997 and 9999, were considered to have no similarity at all to P. fuscovaginae and a further two strains, ICMP11283 and 11284, were considered, by fatty acid analysis, to be more closely related to P. putida. These two strains were related to P. marginalis according to Biolog, and to P. tolaasii according to 16s and rpoB data. The Australian rice isolates were all identified as P. putida Biotype A by fatty acid analysis or as P. asplenii (4 isolates), P. fuscovaginae (2 isolates) or P. fluorescens (1 isolate) by Biolog. Sequencing of the 16s rRNA gene placed the rice isolates with P. fuscovaginae and P. asplenii, whereas rpoB gene sequence analysis showed a higher similarity to P. fuscovaginae than to P. asplenii. This is the first report of P. fuscovaginae in Australia.

Bacteria associated with sheath browning and grain discoloration of rice in East Timor and implications for Australia's biosecurity

A survey of rice growing regions in East Timor (Timor Leste) conducted in 2010 has resulted in the identification of several bacterial species associated with sheath browning and seed discolouration. Bacteria were identified by partial sequencing of the 16s rRNA gene and comparison with sequences from GenBank and EZtaxon databases. Bacterial isolates of known pathogens of rice, corn and cotton were identified, along with non-phytopathogenic endophytes. This is an indication that the disease symptoms are likely to be the result of a pathogen complex rather than caused by a single species. Unfortunately, due to current lack of infrastructure in East Timor, definitive pathogenicity testing of these isolates could not be undertaken. However, it is imperative to increase the capacity of East Timorese to improve their diagnostic skills and management of diseases of rice, and ultimately increase productivity. Furthermore, it is important to heighten the awareness of East Timorese farmers of the importance of diseases in rice production. Evidently, there is a potential source of high risk bacterial pathogens in the rice production system in East Timor and this also poses a risk to Australia’s biosecurity. Potentially devastating pests and diseases of Australian agriculture currently present in Asia could spread into Australia via East Timor. There are a number of economically important diseases of rice that occur in Southeast Asia which do not currently occur in Australia.

Black Scab of Jojoba (Simmondsia chinensis) in Australia Caused by a Putative New Pathotype of Elsinoë australis

A new disease of jojoba in Australia is described. We have demonstrated that this disease is caused by Elsinoë australis, a pathogen which is normally associated with citrus. This pathogen has not been found previously in Australia on citrus or any other crop. The fungus causes a scab on leaves and stems of jojoba and is widely distributed in eastern Australia. Although molecular analysis of the pathogen indicates that it is closely related to the natsudaidai and the sweet orange pathotypes of E. australis, glasshouse and laboratory experiments demonstrate that it is not pathogenic to a range of citrus cultivars grown in Australia. The data indicate that the isolates from jojoba represent a new pathotype of E. australis.

GENOTYPIC ANALYSIS OF MUCOR FROM THE PLATYPUS IN AUSTRALIA

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Genetic diversity within a population of Microlaena stipoides, as revealed by AFLP markers

Microlaena stipoides (Labill.) R.Br. (microlaena), a C3 perennial grass, is common within grazed native pastures in the high-rainfall zone (>550 mm average annual rainfall) of south-eastern Australia. It has the ability to spread via seed production or vegetatively, using both rhizomes and stolons. This experiment aimed to determine how variable a microlaena population was within a single area, with the aim of determining whether microlaena relied on seed or vegetative spread to sustain populations. Leaf samples of microlaena were collected from 85 locations, sampling two transects, within a pasture at Chiltern, in north-eastern Victoria (36°12ʹS, 146°35ʹE). The genetic diversity among samples was analysed using amplified fragment length polymorphism (AFLP) markers. We obtained 1612 fragments, using 10 primers combinations. Polymorphism for the markers ranged from 47% to 65%. These results indicated that the populations of microlaena that exist within the pasture at Chiltern are likely to have undergone some degree of outcrossing (Fst = 0.0219). It is likely that recruitment is occurring from sexual reproduction as well as via clonal spread within the microlaena population examined. This ability to use vegetative spread as well as both sexual and asexual reproduction may make populations of microlaena more resilient in the longer term.